Project Summary: Small RNAs regulate gene expression in a wide range of organisms from yeasts to humans and the fundamental mechanisms governing RNA silencing are conserved across most eukaryotes. The term ?small RNA? encompasses a diverse set of small regulatory RNAs, ranging from ~18-30 nucleotides in length, all bound by members of the Argonaute protein family. The first small RNAs identified were microRNAs, discovered in C. elegans as regulators of development timing (Ambros and Ruvkun labs). Several years later, the phenomenon of RNA interference (RNAi), gene silencing induced by the introduction of double stranded RNA, was also discovered in C. elegans (Fire and Mello labs). Much work since then has uncovered the roles of a diverse set of proteins that regulate genes by way of small RNAs at the level of transcription, translation, and mRNA stability. By regulating both endogenous and foreign RNAs, small RNAs play a critical role in development, genome stability, and viral defense. The goal of this research program is to investigate the mechanisms by which RNA silencing pathways modulate gene expression and maintain genome integrity. Like Ambros, Ruvkun, Fire, and Mello, we will use the nematode, C. elegans, as a model system to study RNA silencing because worms combine the best of genetic, cytological, molecular, and biochemical tools. There are many fundamental unanswered questions in our understanding of RNA silencing that we plan to address in this proposal. We and others have determined the localization for many factors in the RNA silencing pathway but there has been little to no cytological exploration the RNA and DNA components. Furthermore, the mechanism by which targeted transcripts are recognized and routed into the RNA silencing pathways is unknown. Our first goal is to localize the mRNAs targeted by RNA silencing and to determine the protein requirements for their physical movement from transcription in the nucleus to turnover in the cytoplasmic RNA silencing compartment, the Mutator foci. We will further examine whether RNA silencing affects the subnuclear positioning of the targeted gene loci, to promote mRNA localization and RNA silencing. Despite much work in the field, there are proteins attributed to small RNA pathway for which the mechanism of action is unknown. We also believe there are components of the pathway that have yet to be identified. Our second goal is to use a combination of molecular biology and cytology to examine uncharacterized components of the pathway in the processes of recruitment and retention of RNA in the Mutator foci and in modification of mRNA targets. Our third goal is to identify and characterize new components of the RNA silencing complex using proteomics. This research will shed light on conserved pathways that regulate gene expression and are critical for development, antiviral immunity, and genome organization.